MYCN protein expression level in neuroblastoma patients predicts prognosis better

Charvy Rana, Department of Pathology, AIIMS, New Delhi

What is MYCN protein?

Cancer often involves the inactivation of tumor-suppressor genes and/or over-activation of oncogenes. The MYCN proto-oncogene is one such example, implicated for its amplified or “oncogenic” status in several neuronal and non-neuronal cancers (those of lung, ovaries, prostate, and more)¹. Normally expressed during the early development of the neural crest cells, MYCN codes for a transcription regulator, MYCN. The basic helix-loop-helix-Zip (bHLH-Zip) domain at the C-terminus of MYCN endows it the ability to bind to DNA as a heterodimer with another bHLH-containing protein, MAX. Whereas the transactivation domain at N-terminus enables its interactions with nuclear proteins, mediating both activation and repression of target genes².

MYCN as a biomarker in neuroblastoma

Amplification of the MYCN oncogene is an established marker for high-grade cases of neuroblastoma, a pediatric solid tumor, which has a higher risk of metastasis and poor prognosis. Neuroblastoma is a type of cancer that usually develops in immature nerve cells in the proximity of the adrenal glands.

Current clinical practices focus on MYCN at the gene or transcript level, using methods based on primer amplification (e.g. PCR, qPCR, multiplex ligation-dependent probe amplification-MLPA) or probe hybridization (e.g. FISH, CISH). Of these, fluorescence in situ hybridization (FISH) is considered the “gold standard” for risk group stratification of neuroblastoma patients based on the MYCN status. However, researchers have shown the over-expression of MYCN protein in tumors with normal MYCN copy numbers and no expression in tumors carrying amplified MYCN gene.

The lack of reliable MYCN-specific antibodies has restricted the detection of MYCN protein through immunohistochemistry (IHC) in hospitals and research. The MYCN antibodies tested so far have displayed substandard specificity and sensitivity, compared to FISH results. In a recent study, researchers tested out two antibodies for MYCN IHC: one chosen by pathologists at SCMC (#51705, Cell Signaling Technology) after trying out different MYCN antibodies, and the second (#84406 s, Cell Signaling Technology), a commercial antibody never used for IHC before. They established a reliable method to detect MYCN protein which correlated with neuroblastoma patient outcomes.

Assessing the IHC-based detection of MYCN

1. First, the accuracy of the FISH procedure was confirmed by comparing the results with those of whole exome sequencing. The survival curve analysis of MYCN amplified (FISH⁺) with MYCN non-amplified (FISH^–) across the different INSS stages highlighted the significance of the FISH test in predicting the poor outcome of the only stage 3 patients while failing to detect the more adverse, INSS stage 4 patients.
2. On comparison of the IHC results of the first antibody (#51705), an agreement of around 40% with the FISH results was observed, whereas the second antibody (#84406 s) could correctly detect MYCN status with over 80% accuracy. The non-concordant (~20%) cases were observed to be associated with clinical outcomes rather than gene status.
3. The event-free survival (EFS) rates calculated for cases as per their IHC scores within the MYCN amplified and non-amplified groups were significantly different (p-value<0.05) than the EFS rates contrasted solely based on their FISH amplification status. Combining the FISH and IHC results gave the best prognostic estimate for all the cases.
4. The IHC results were further tested by the detection of the MYCN protein expression in a subset of samples through western blotting. The MYCN protein level coincided with the IHC results, although not with the mRNA expression level. However, transcripts of proteins promoting MYCN protein stability and those involved in MYCN degradation were upregulated and downregulated respectively, consistent with the IHC results.

Conclusion

This study confirms the utility of a commercial antibody (84406 s, Cell Signaling Technology) to quantify the amount of stable MYCN protein in neuroblastoma cases. IHC results obtained with this antibody correspond well with disease prognosis and staging. Moreover, the observation of compatible trends in the expression of proteins regulating the stability of MYCN with the IHC results implicates the role of post-transcriptional and post-translational modifications in influencing the active MYCN protein level.

Thus, MYCN IHC is a more reliable indicator of the biologically more relevant MYCN protein. A conjoint report of MYCN gene and protein status performed through FISH and IHC could better predict the clinical outcome in neuroblastoma patients.

Abbreviations used:

PCR- Polymerase chain reaction, qPCR- Quantitative real-time PCR, MLPA- Multiplex ligation-dependent probe amplification, CISH- Chromogenic in situ hybridization, FISH: Fluorescence in situ hybridization, IHC- Immunohistochemistry, SCMC- Shanghai Children’s Medical Center, EFS- event-free survival, INSS- International Neuroblastoma Staging System.

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References:

Yang, Y., Zhao, J., Zhang, Y., Feng, T., Yv, B., Wang, J., Gao, Y., Yin, M., Tang, J., & Li, Y. (2022). MYCN protein stability is a better prognostic indicator in neuroblastoma. BMC pediatrics, 22(1), 404. https://doi.org/10.1186/s12887-022-03449-1

Other supporting references used in the article:

1. Liu, R., Shi, P., Wang, Z., Yuan, C., & Cui, H. (2021). Molecular Mechanisms of MYCN Dysregulation in Cancers. Frontiers in oncology, 10, 625332. https://doi.org/10.3389/fonc.2020.625332
2. Cheung, L., E., J., Haber, M., & D., M. (2013). The MYCN oncogene. In Y. Siregar (Ed.), Oncogene and Cancer—From Bench to Clinic. InTech. https://doi.org/10.5772/54813

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